The initiation of the immune response involves a complex array of intercellular signals. These signals typically involve soluble cytokines coupled with a number of cell--cell contact dependent signals. The contact dependent events, most notably activation of the T-cell receptor, lend specificity to the response whereas the soluble mediators are generally responsible for maintenance of cell differentiation and proliferation. Tumor Necrosis Factor (TNF) and LT-.alpha. are two polypeptides generally recognized for involvement with the initiation of the immune response.
TNF and LT-.alpha. are soluble proteins noted originally for their ability to inhibit the growth of tumors. [L. Old, "Tumor Necrosis Factor," Science, 230, 630 (1985)]. Further research demonstrated that both proteins exhibit a wide range of activities. TNF is synthesized in response to various inflammatory insults by a variety of cell types including both hematopoietic and nonhematopoietic cells, while LT-.alpha., in contrast, is made specifically by lymphocytes. The two known TNF receptors do not appear to discriminate between LT-.alpha. and TNF. [T. Schall et al., "Molecular Cloning and Expression of a Receptor for Human Tumor Necrosis Factor," Cell, 61, 361-370 (1990); C. Smith et al., "A Receptor for Tumor Necrosis Factor Defines an Unusual Family of Cellular and Vital Proteins," Science 248, 1019 (1990)]. In general, LT-.alpha. and TNF display similar spectra of activities in in Vitro systems, although LT-.alpha. is often less potent. [J. Browning et al., Studies of The Differing Affects Of Tumor Necrosis Factor And Lymphotoxin On The Growth of several Human Tumor Lines," J. Immunol., 143, 1859 (1989)].
TNF appears to play a major role in specific aspects of metabolic control, in the response to endotoxin shock, and in the regulation of hematopoietic cell development. [B. Beutler et al., "The History, Properties, and Biological Effects of Cachectin," Biochemistry, 27, (1988); M. Akashi et al., "Lymphotoxin: Stimulation And Regulation of Colony Stimulating Factors in Fibroblasts," Blood, 74, 2383 (1989); G. Roodman et al., "Tumor Necrosis Factor-alpha and Hematopoietic Progenitors: Effects Of Tumor Necrosis Factor On The Growth of Erythroid Progenitors CFU-E And BFU-E And The Hematopoietic Cell Lines k562, HL60, And HEL Cells," Exp. Hematol., 15, 928 (1987)].
Along with IL-1 and IL-6, TNF is also a major mediator of the inflammatory response. [D. Cavender et al., "Endothelial Cell Activation Induced By Tumor Necrosis Factor And Lymphotoxin," Amer. Jour. Path., 134, 551 (1989); R. Cotran et al., "Endothelial Activation Its Role In Inflammatory And Immune Reactions," in Endothelial Cell Biology, (Plenum Press, Simonescu & Simonescu, eds., 1988) 335]. TNF also appears to be involved in T cell activation under certain conditions. [M. Shalaby et al., "The Involvement Of Human Tumor Necrosis Factors-.alpha. And-.beta. In The Mixed Lymphocyte Reaction," J. Immunol., 141, 499 (1988); N. Damle et al., "Distinct Regulatory Effects of IL-4 and TNF-.alpha. During CD3-Dependent and CD3-Independent Initiation Of Human T-Cell Activation," Lymph. Res., 8, 85 (1989); G. Ranges et al., "Tumor Necrosis Factor-.alpha. As A Proliferative Signal For An IL-2-Dependent T Cell Line: Strict Species Specificity of Action," Amer. Assoc. Immunol., 142, 1203 (1989); G. Ranges et al., "Tumor Necrosis Factor .alpha./Cachectin Is A Growth Factor For Thymocytes," J. Exp. Med., 167, 1472 (1988); P. Scheurich et al., "Immunoregulatory Activity Of Recombinant Human Tumor Necrosis Factor (TNF)-.alpha.: Induction Of TNF Receptors On Human T Cells And TNF-.alpha.-Mediated Enhancement Of T Cell Responses," J. Immunol., 138, 1786 (1987)].
TNF is produced by several types of cells, including monocytes, fibroblasts, T cells and Natural Killer (NK) cells. [D. Goeddel et al., "Tumor Necrosis Factors: Gene Structure And Biological Activities," Cold Spring Harbor Symposium Quant. Biol., 51, 597 (1986); D. Spriggs et al., "Tumor Necrosis Factor Expression In Human Epithelial Tumor Cell Lines," J. Clin. Invest., 81, 455 (1988); M. Turner et al., "Human T cells From Autoimmune and Normal Individuals Can Produce Tumor Necrosis Factor," Eur. J. Immunol., 17, 1807 (1987)]. Investigators have also detected murine and human forms of TNF that are associated with the surface of various cells either as a transmembrane protein or a receptor-bound molecule. [B. Luettig et al., "Evidence For the Existence of Two Forms of Membrane Tumor Necrosis Factor: An Integral Protein and a Molecule Attached To Its Receptor," J. Immunol., 143, 4034 (1989); M. Kriegler et al., "A Novel Form of TNF/Cachectin Is a Cell Surface Cytotoxic Transmembrane Protein: Ramifications For the Complex Physiology of TNF," Cell, 53, pp. 45-53 (1988); and M. Kinkhabwala et al., "A Novel Addition To the T Cell Repertory," J. Exp. Med., 171, pp. 941-946 (1990)].
LT-.alpha. also has many activities, generally similar, but not identical to those of TNF, including tumor necrosis, induction of an antiviral state, activation of polymorphonuclear leukocytes, induction of class I major histocompatibility complex antigens on endothelial cells, induction of adhesion molecules on endothelium and growth hormone stimulation. [N. Ruddle and R. Homer, "The Role of Lymphotoxin in Inflammation," Prog. Allergy, 40, pp. 162-182 (1988)]. Both LT-.alpha. and TNF are ligands to members of the nerve growth factor (NGF) receptor family. [S. Mallett and A. N. Barclay, "A New Superfamily Of Cell Surface Proteins Related To The Nerve Growth Factor Receptor," Immunology Today, 12, 7, 220-223 (1991).]
In contrast to TNF, LT-.alpha. secretion appears to be a specific property of only activated T cells and certain B-lymphoblastoid tumors. [N. Paul et al., "Lymphotoxin," Ann. Rev. Immunol., 6, 407 (1988)]. Some researchers have also indicated that a membrane-associated form of LT-.alpha. may be expressed on the surface of lymphocytes under certain circumstances [J. Hiserodt, et al., "Identification of Membrane-Associated Lymphotoxin (LT) On Mitogen-Activated Human Lymphocytes Using Heterologous Anti-LT Antisera In Vitro," Cell. Immunol., 34, pp. 326-339 (1977); C. Ware et al., "Mechanisms of Lymphocyte-Mediated Cytotoxicity," J. Immunol., 126, pp. 1927-1933 (1981); U. Anderson et al. J. Immunol. Methods, 123, 233 (1989); Y. Abe et al., Jpn. J. Canc. Res., 82, 23 (1991); Y. Abe et al., "Studies of Membrane Associated and Soluble (Secreted) Lymphotoxin In Human Lymphokine-Activated T-Killer Cells In Vitro," Lymphokine and Cytokine Research, 11, 2, 115-121 (1992)].
In recent years genes for both TNF and LT-.alpha. have been isolated and cloned, leading to their complete characterization and to the availability of recombinant forms of both proteins. [P. Gray et al., "Cloning and Expression of cDNA For Human Lymphotoxin, A Lymphokine With Tumor Necrosis Activity," Nature, 312, pp. 121-124 (1984); D. Pennica et al., "Human Tumor Necrosis Factor: Precusor Structure, Expression And Homology To Lymphotoxin," Nature, 312, 724 (1984)].
Other "cytokine-like" cell surface proteins including the CD40 protein have recently been shown to share certain similarities with TNF and LT-.alpha.. Like TNF and LT-.alpha., the CD40 protein is a ligand to members of the TNF/nerve growth factor (NGF) receptor family. [S. Mallett and N. Barclay, Immunology Today, 12, pp. 220-223 (1991)]. The CD40 protein is a 277-amino acid protein expressed on the surface of B lymphocytes, epithelial cells, and some carcinoma cell lines. [R. Armitage et al., Nature, 357, pp. 80-82 (1992); T. Farrah and C. Smith, "Emerging Cytokine Family," Nature, 358, p. 26 (1992)].
We have now identified a novel surface protein, lymphotoxin-.beta. (LT-.beta.) or p33. LT-.beta. has been identified on the surface of several types of lymphocyte cells, including OKT3-stimulated primary T cells, antigen specific IL-2 dependent CTL clones, and a PMA-stimulated human T cell hybridoma II-23.D7. LT-.beta. targets LT-.alpha. produced in the cell to the cell membrane where LT-.beta. and LT-.alpha. appear as a complex (designated "LT-.alpha./LT-.beta." throughout this disclosure). The LT-.alpha./LT-.beta. complex is believed to be a novel mechanism for membrane expression of LT-.alpha. by activated T-cells. [Androlewicz et al., "Lymphotoxin Is Expressed As a Heteromeric complex With A Distinct 33 kDa Glycoprotein On The Surface Of An Activated Human T Cell Hybridoma," Journal Of Biological Chemistry, 267, pp. 2542-2547 (1992)]. The LT-.alpha./LT-.beta. complex may exhibit cytolytic and cell regulatory activity similar to the soluble LT-.alpha., TNF and CD40 proteins. The membrane-associated LT-.beta. complexed with LT-.alpha. may represent, as a complex, a novel ligand for T cell interactions with other cells and may also be useful in targeted cell lysis.